Dan
The main problem is that FITC is detected in FL2 AND FL3 and you can't
compensate FL1 vs FL3 directly. (You can with Verity Software House's WinList,
but I don't have this!) You can adjust the compensation through FL2 by using
all compensation settings.
I use the FACStar+ where I can put a better filter in FL3, 630 nM bandpass,
instead of the 650 nM longpass that is in our FACScan.
Are you setting the compensation values with SINGLE stained samples?
And yes, we all have seen the dim PI positives. I use as litte PI as possible
(0.5 micrograms per ml) but still have seen them. I've heard some people WASH
after PI staining, but the A-V protocols are supposed to be no wash!
Dennis
______________________________ Reply Separator _________________________________
Subject: PI vs. 7-AAD Nuclear Staining
Author: DJS at allp.com at @UCSD
Date: 3/15/96 10:24 AM
I have been trying to surface stain apopotic MCF-7 breast cancer cells with
Annexin V-FITC and stain the DNA with PI to test for necrosis vs apoptosis.
Even though the emission spectra shouldn't overlap I am having to
compensate for what appears to be overlap in both FL1 and FL3.
Also the PI appears to be passing through the membrane of viable cells.
Any suggestions on either problem (Compensation or viability stain)
Would switching to 7-AAD be a possibility.
Thanks for the input,
Dan Smith
San Diego
djs at allp.com
>-- Saved internet headers (useful for debugging)
>Received: from flowcyt.cyto.purdue.edu by mail.ucsd.edu; id OAA22599 sendmail 8
>Received: by flowcyt.cyto.purdue.edu (950215.SGI.8.6.10/930416.SGI.AUTO) for cy
>Received: from ALLP.COM by flowcyt.cyto.purdue.edu via ESMTP (950215.SGI.8.6.10
>Received: from msmail.allp.com (MSMAIL.ALLP.COM [205.185.36.11]) by ALLP.COM (8
>Received: by msmail.allp.com with Microsoft Mail id <3149B5F8 at msmail.allp.com>;
>From: Dan Smith <DJS at allp.com>
>To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>Subject: PI vs. 7-AAD Nuclear Staining
>Date: Fri, 15 Mar 96 10:24:00 PST
>Message-ID: <3149B5F8 at msmail.allp.com>
>Encoding: 12 TEXT
>X-Mailer: Microsoft Mail V3.0
>X-PMFLAGS: 33554560 0